Project Summary/Abstract C. difficile infection (CDI) is the most common cause of infectious diarrhea in the healthcare setting with about 453,000 cases and 29,000 deaths yearly in the U.S. as reported by CDC in 2015. A continual rise in severe CDI has been observed worldwide. Currently, standard treatment for CDI is the administration of antibiotics. While effective, these treatments do not prevent and may contribute to a disease recurrence rate of 15-35%. Prevention of recurrence is one of the most challenging aspects in the field. CDI symptoms range from diarrhea to intestinal inflammation/lesion and death. Symptoms are mainly caused by two exotoxins TcdA and TcdB. Both toxins share a very similar domain structure including the N-terminal catalytic glucosyltransferase domain (GT), the autolytic cysteine proteinase domain (CPD), the central translocation domain (TM), and the C-terminal receptor-binding domain (RBD). No vaccine against CDI is currently licensed. The objective of this project is to develop novel mucosal vaccines that target both C. difficile toxins and colonization/adhesion factors. We have previously demonstrated effective protection with an earlier immunogen mTcd138, which contains the GT and CPD domains of TcdB and the RBD of TcdA. We further enhanced mTcd138 by: 1) adding the RBD of TcdB to mTcd138, resulting in fusion protein mTcd169; 2) fusing mTcd169 with Salmonella typhimurium flagellin (sFliC), resulting in fusion protein Tcd169Fl; and 3) expressing mTcd138 in non-toxigenic C. difficile strains, designated as NTCD_Tcd138. Oral immunizations with NTCD_Tcd138 spores provided full protection in mice and significant protection in hamsters against challenge with hyper-virulent C. difficile strains. In addition, we expressed and purified C. difficile protein Cwp84, which is a cysteine protease and plays a critical role in maturation of surface-layer proteins. It was reported that Immunization with Cwp84 provides significant protection in hamsters by delaying C. difficile colonization. In this project, we will: 1) further express mTcd169 and Tcd169Fl in non-toxigenic C. difficile 85 (NTCD) to generate strains NTCD_Tcd169 and NTCD_Tcd169FL, respectively; 2) evaluate the following four immunogens as potential vaccine candidates in mice and hamster models of CDI: Tcd169Fl, a mixture of Tcd169Fl and Cwp84 (designated as Tcd169Fl/Cw), NTCD_Tcd169, and NTCD_Tcd169Fl.